卫氏并殖吸虫感染及白细胞介素2对小鼠细胞免疫功能影响的观察

本文以抗小鼠Ｌ３Ｔ４、Ｌｙｔ２单克隆抗体检测小鼠脾细胞中ＴＨ、Ｔｓ数，３Ｈ－ＴｄＲ掺入法淋巴细胞转化试验及白细胞介素２活性测定的方法，对经口接种卫氏并殖吸虫囊蚴小鼠及感染此虫并接受白细胞介素２治疗的小鼠进行了细胞免疫功能的动态观察。结果显示，感染小鼠脾细胞中ＴＨ减少、Ｔｓ增多，ＴＨ／Ｔｓ比值降低。刀豆素蛋白Ａ诱导的淋巴细胞转化受抑制。白细胞介素２诱导及活性测定中，靶细胞掺入的ｃｐｍ值降低。提示卫氏并殖吸虫的感染可使小鼠细胞免疫功能紊乱、降低。接受白细胞介素２活性水平高于感染组，接近正常对照组；提示白细胞介素２对增强感染卫氏并殖吸虫的小鼠细胞免疫功能有一定作用。     

血吸虫感染新模型小鼠细胞因子的诱生实验

目的　在已建立了一种血吸虫感染伴随免疫新模型的基础上 ,检测该模型小鼠脾细胞培养诱生白细胞介素 -2 (IL-2 )和白细胞介素 -5(IL-5)的水平 ,初步探讨该新模型抗再感染的细胞免疫机制。方法　小鼠感染日本血吸虫尾蚴 2 0 d后 ,按 3 0 0 mg/ (kg· d)腹腔注射酚酶抑制剂——丙烯基硫脲 ,同时设立未用药对照组和正常小鼠组 ,感染后第 42 d剖杀动物 ,分别用成虫抗原 (SWAP)、虫卵抗原 (SEA)及刀豆素 A(Con A)体外刺激脾细胞诱生细胞因子 ,并检测 IL -2和 IL -5的诱生水平。结果　新模型小鼠经 SWAP诱生 IL -2的水平高于未用药对照组和正常组小鼠 ;经 SEA诱生 IL-2的水平低于对照组小鼠 ,而与正常组小鼠无差异 ;经 Con A诱生 IL-2的水平与对照组小鼠和正常小鼠无差异。此外 ,新模型小鼠经 SWAP、SEA及 Con A 3种抗原刺激诱生 IL -5的水平均明显低于对照组和正常组小鼠。结论　伴随免疫新模型小鼠体内成虫诱导的细胞免疫似以 Th1细胞功能占优势 ,而 Th2细胞功能被抑制     

约氏疟原虫红内期感染小鼠的免疫反应及IL－2、IFN－γ免疫调节作用的研究

在原虫血症上升期，致死株约氏疟原虫（Ｐ，ｙ，）感染鼠脾细胞的增殖反应受到抑制，无γ－干扰素（ＩＦＮ－γ）产生，血清中未产生特异抗体，仅产生与正常鼠相同的白细胞介素２（ＩＬ－２０）；与此不同，非致死株Ｐ．ｙ．感染鼠脾细胞增殖反应增强，产生ＩＦＮ－γ及更多的ＩＬ－２，血清中还无特异抗体产生。在原虫血症下降期，脾细胞的增殖反应升高，较上升期有更多的ＩＬ－２及ＩＦＮ－γ生成，体内血清中出现滴度较高的特异抗体。免疫鼠脾细胞的增殖反应较前两者明显升高，体内血清特异抗体水平亦明显上升，ＩＬ－２及ＩＦＮ－γ水平与感染鼠原虫下降期相同。表明在Ｐ．ｙ．感染小鼠中，致死株的初次感染可抑制细胞免疫，非致死株在原虫上升期即可出现细胞免疫。原虫下降期细胞免疫功能进一步增强，高水平抗体生成在清除原虫中可能起重要作用     

尿酸辅助树突细胞免疫对小鼠淋巴细胞增殖及CTL效应的影响

目的：观察尿酸辅助HBsAg蛋白负载的树突细胞免疫接种对小鼠免疫功能的影响。方法：将负载HB-sAg的小鼠骨髓来源树突细胞经尾静脉注射接种小鼠，1次／w，共2次。分DC（树突状细胞）对照组、联合尿酸组、尿酸对照组。MTT法检测体外经HBsAg或PBS重刺激的脾T淋巴细胞增殖反应；流式细胞仪法检测CTL（细胞毒性T淋巴细胞）活性。结果：免疫2周时，小鼠脾T细胞增殖反应及体内HBsAg特异性CTL的杀伤活性，联合尿酸组明显强于各对照组。结论：尿酸可促进负载HBsAg树突细胞免疫后小鼠脾T淋巴细胞的增殖及HBsAg特异性CTL效应。尿酸具有增强DC疫苗免疫效应的活性，可用作研制抗HBV的治疗性疫苗的免疫佐剂。     

紫外线减毒日本血吸虫尾蚴免疫小鼠的细胞免疫应答和细胞因子的动态变化

目的 :检测细胞免疫在血吸虫疫苗保护性免疫中的作用。方法 :用紫外线减毒日本血吸虫尾蚴 30 0± 5条免疫小鼠及日本血吸虫尾蚴 2 5± 3条感染小鼠。于第 2 wk、4 wk、8wk和 12 wk分别用血吸虫成虫抗原 ( SWAP)、虫卵抗原 ( SEA )及丝裂原 ( Con A或 L PS)体外刺激脾细胞和腹腔巨噬细胞 ( M) ,观察脾淋巴细胞的增殖反应及 M产生 IL- 1和脾细胞产生 IL- 2的活性的动态变化。结果 :两组鼠的脾细胞于免疫或感染后 2 wk- 8wk经 SWAP或 SEA刺激 T淋巴细胞增殖反应显著增强 ,第 12 wk呈现明显抑制 ;免疫组的 M和脾细胞经 SWAP或 SEA刺激于接种后第 4wk IL- 1和 IL- 2活性均显著增高 ,感染组的 M和脾细胞经 SWAP刺激 IL- 1于第 8wk- 12 wk活性增高、IL- 2于第 12 wk活性增高。结论 :提示减毒尾蚴免疫接种能较早地激活 T细胞增殖和细胞因子产生 ,在血吸虫保护性免疫中起重要作用。     

柯萨奇3m病毒和T-2毒素致小鼠慢性心肌损伤细胞免疫功能改变

目的 观察慢性心肌损伤过程中小鼠细胞免疫功能的改变。方法 BALB／C小鼠随机分成4组，病毒（柯萨奇3m，CVB3m）组、毒素（T-2）＋病毒组、毒素组和对照组。观察各组小鼠脾淋巴细胞亚群及其在刀豆蛋白A（CoA）刺激下增殖及白细胞介素-2（IL-2）的分泌水平。结果 毒素＋病毒组小鼠心肌形成慢性损伤，感染病毒7d时，毒素＋病毒组脾淋巴细胞亚群、淋巴细胞增殖反应及IL-2的分泌水平均低于病毒组，14d后虽有不同程度的提高，但低于病毒组最高值。结论 T-2毒素染毒小鼠感染CVB3m后，心肌出现慢性损伤，这可能是由于淋巴细胞数量、功能和活性异常，细胞免疫功能紊乱，导致发病高峰延迟，阻碍早期清除病毒及杀伤受染细胞的作用有直接关系。     

紫外线减毒日本血吸虫尾蚴免疫小鼠的细胞免疫应答和细胞因子的动态变化

目的 :检测细胞免疫在血吸虫疫苗保护性免疫中的作用。方法 :用紫外线减毒日本血吸虫尾蚴 300±5条免疫小鼠及日本血吸虫尾蚴 25±3条感染小鼠。于第 2wk、4wk、8wk和 12wk分别用血吸虫成虫抗原(SWAP)、虫卵抗原 (SEA)及丝裂原(ConA或LPS)体外刺激脾细胞和腹腔巨噬细胞(Mφ),观察脾淋巴细胞的增殖反应及 Mφ产生 IL- 1和脾细胞产生 IL- 2的活性的动态变化。结果 :两组鼠的脾细胞于免疫或感染后2wk- 8wk经 SWAP或 SEA刺激 T淋巴细胞增殖反应显著增强,第12wk呈现明显抑制;免疫组的Mφ和脾细胞经SWAP或SEA 刺激于接种后第4wk IL-1 和IL-2 活性均显著增高, 感染组的Mφ和脾细胞经SWAP 刺激IL-1 于第8wk-12wk活性增高、IL-2 于第12wk 活性增高。结论: 提示减毒尾蚴免疫接种能较早地激活T 细胞增殖和细胞因子产生, 在血吸虫保护性免疫中起重要作用。     

IL-15表达质粒联合HPV基因疫苗诱导细胞免疫应答的实验研究

目的研究白细胞介素15(IL-15)真核表达质粒,对人乳头瘤病毒(HPV)16E7基因疫苗所诱导的小鼠特异性细胞免疫应答的影响。方法构建含IL-15的真核表达质粒pcDNA3.1-IL-15。将该质粒与HPV16 E7基因疫苗通过肌肉注射方式免疫雌性BALB/c小鼠。基因免疫后测定其血清γ-干扰素(IFN-γ)水平;并制备脾淋巴细胞悬液,经体外E7蛋白再刺激后用MTT法检测其T淋巴细胞增殖情况。结果pcDNA3.1-IL-15与pcD-NA3.1-E7共同注射,可以提高免疫小鼠血清中IFN-γ水平至414.1pg/ml,与E7 空质粒组、E7组比较差异有统计学意义(P<0.01)。pcDNA3-1-IL-15与pcDNA3.1-E7共同注射,可以增强特异性T细胞增殖反应,OD570差值为1.313,与其他各组比较差异均有统计学意义(P<0.01)。结论IL-15真核表达质粒可以提高HPV16 E7基因疫苗的免疫原性,增强小鼠细胞免疫应答。     

弓形虫亲环蛋白亚单位疫苗的免疫保护性研究

目的研究弓形虫亲环蛋白(Cyclophilin,CyP)亚单位疫苗的免疫保护性。方法 30只BALB/c小鼠随机分为3组:实验组、佐剂组和PBS对照组,分别肌注pET-28a-TgCyP重组蛋白100μg/只、佐剂100 ul/只和PBS 100 ul/只,共免疫3次,每次间隔2周。末次免疫后2周,各组分别取4只小鼠处死,取脾脏,检测CD4+、CD8+及细胞因子。同时,采用阿尔玛蓝细胞增殖与细胞毒性检测试剂测定小鼠脾淋巴细胞的增殖应答。其余小鼠腹腔攻击感染Rh株弓形虫速殖子500个,观察其存活情况。结果 pET-28a-TgCyP重组蛋白亚单位疫苗能诱发小鼠产生细胞免疫和体液免疫反应,小鼠的CD4+T细胞(62.59±4.17)%、CD8+T细胞(8.53±0.46)%与PBS及佐剂对照组比较显著增殖(P<0.01),其脾细胞培养液白细胞介素-2(IL-2)、γ干扰素(IFN-γ)显著升高,小鼠脾淋巴细胞增殖应答显著增强(P<0.01)。弓形虫攻击感染216 h后,实验组小鼠免疫保护率为33.3%,佐剂对照组及PBS对照组小鼠均全部死亡。结论 pET-28a-TgCyP重组蛋白亚单位疫苗具有较强的免疫原性,能...     

卫氏并殖吸虫感染及白细胞介素2对小鼠细胞免疫功能影…

本文以抗小鼠Ｌ３Ｔ４、Ｌｙｔ２单克隆抗体检测小鼠脾细胞中ＴＨ、ＴＳ数，＾３Ｈ－ＴｄＲ掺入法淋巴细胞转化试验及白细胞介素２活性测定的方法，对经口接种卫氏并殖吸虫囊蚴小鼠及感染此虫并接受白细胞介素２治疗的小鼠进行了细胞免疫功能的动态观察。结果显示，感染小鼠脾细胞中ＴＨ减少、ＴＳ增多，ＴＨ／ＴＳ比值降低。刀豆素蛋白Ａ诱导的淋巴细胞转化受抑制。白细胞介素２诱导及活性测定中，靶细胞掺入的ｃｐｍ值降低。提示卫     

Lack of CXCR3 delays the development of hepatic inflammation but does not impair resistance to Leishmania donovani

CXC chemokine receptor 3 (CXCR3) ligands CXCL9 and CXCL10 are produced at high levels in mice and humans infected with Leishmania donovani, but their contribution to host resistance against L. donovani is not clear. Here, using CXCR3(-/-) mice, we demonstrate that, although CXCR3 regulates early immune cell trafficking and hepatic inflammation during L. donovani infection, it is not essential for immunity against L. donovani, unlike L. major. CXCR3(-/-) C57BL/6 mice show a delayed onset of hepatic inflammation and granuloma formation after L. donovani infection. However, they mount an efficient T helper cell type 1 response, recruit T cells to the liver, and control parasite growth as efficiently as do CXCR3(+/+) C57BL/6 mice.     

Cross-protection against Leishmania donovani but not L. Braziliensis caused by vaccination with L. Major soluble promastigote exogenous antigens in BALB/c mice

Vaccinating with soluble Leishmania major promastigote exogenous antigens (LmSEAgs) protects mice against challenge with L. major. To explore the potential of LmSEAgs to cross-protect against infection with other species of Leishmania, BALB/c mice were immunized with LmSEAgs prior to challenge with either L. donovani or L. braziliensis promastigotes. Such mice were protected against L. donovani but not L. braziliensis infection. Leishmania braziliensis-infected mice developed lesions that were not significantly different from those of controls and that contained 13-fold more parasites. In contrast, immunized mice infected with L. donovani were protected as illustrated by low splenic parasite loads (as much as 4,913-fold fewer parasites). This protection corresponded to significant increases in gamma interferon and low production of interleukin-4 (IL-4) IL-4 or IL-10, which suggested an enhanced type 1 response.     

Human placental extract offers protection against experimental visceral leishmaniasis: a pilot study for a phase-I clinical trial

An aqueous extract of human placenta (HPE) was found to offer protection against established experimental visceral leishmaniasis in BALB/c mice and hamsters, whether the Leishmania donovani strain involved was one that was sensitive or resistant to pentavalent antimony. Intraperitoneal administration of the extract, into mice or hamsters that had been infected 2 months previously, led to antileishmanial T-cell proliferation among splenic mononuclear cells, the generation of host-protective cytokines (interferon-gamma, tumour necrosis factor and interleukin-12) and the upregulation of the expression of inducible nitric oxide synthase (and subsequent NO generation) in splenocytes. Furthermore, splenic macrophages from the HPE-treated mice showed increased generation of reactive oxygen species and enhanced surface expression of antigens of major histocompatibility complex class II (MHCII), and the extract restored the otherwise-defective antigen-presenting ability of the macrophages. Thus, in mice and hamsters infected with L. donovani, HPE therapy can stimulate both arms of the host's immune system and favour the complete resolution of the leishmanial infection. Among five human cases of visceral leishmaniasis, 30 daily intramuscular injections of HPE, at doses much lower than those used in the experimental infections, also gave very promising results. Based on the results of this pilot study, a further evaluation of the efficacy of HPE therapy, which may offer a cost-effective way of improving the treatment of antimony-resistant cases of visceral leishmaniasis, is being undertaken.     

Immunosuppression associated with visceral leishmaniasis of hamsters

Immunosuppression was demonstrated during the course of Leishmania donovani infection of outbred and inbred hamsters. Proliferative responses of splenic lymphocytes to the mitogen concanavalin A (Con A) and to promastigote antigens were used as indicators of immune responsiveness. Although splenic lymphocyte proliferative responses to parasite antigens were demonstrable 3 weeks after challenge, antigen specific lymphocyte responses diminished as the infection progressed. Two types of immunosuppression were demonstrable. The first was a non-specific anergy of splenic lymphocytes to Con A stimulation. Thus, spleen cells from infected animals did not actively suppress the Con A responses of normal lymphocytes in mixed cultures A second immunosuppression mechanism, specific for leishmania antigens was mediated by a nylon wool non-adherent cell population. The suppressor, tentatively identified as a T cell population, inhibited the proliferation of parasite antigen sensitized responder lymphocytes in mixed culture. Elimination of the parasite burden by glucantime therapy restored responsiveness of lymphocytes to parasite antigens. Con A responses, however, remained suppressed 1 week after drug cure.     

Vectorial efficacy of Phlebotomus argentipes in Kala-azar endemic foci of Bihar (India) under natural and artificial conditions

Ability of Phlebotomus argentipes to acquire Leishmania donovani the causative agent of Indian Kala-azar was evaluated in the laboratory. Flies were fed artificially on infected blood suspensions, using a chick-skin-membrane feeding apparatus, and naturally on Leishmania donovani infected mice. In addition flies collected from different endemic areas were dissected and examined for natural infection. Flies fed on infected mice showed significantly higher feeding rate (14.4%, p < 0.01) compared to that of other experiments (9%, 8.75%) but the percentage of infection was very low (2.43%). No Chi-square comparison was made between infection rate and feeding rate because of low value in infection rate (less than 5). Flies dissected for natural infection showed only 0.1% infection. Not much difference was observed in the intensity of Leishmania donovani infection in the mid gut of sandflies examined from any of these experiments. These observations have confirmed that Phlebotomus argentipes has ability to acquire infection and it provides the final piece of evidence that Phlebotomus argentipes is the vector of Leishmania donovani in Bihar State.     

Selective inability of spleen antigen presenting cells from Leishmania donovani infected hamsters to mediate specific T cell proliferation to parasite antigens

Golden hamsters (Mesocricetus auratus) infected with Leishmania donovani develop a disease similar to human kala-azar. There is conspicuous hypergammaglobulinaemia and their T cells do not respond to stimulation by parasite antigens. The impairment of the cellular immune response seems to be restricted to parasite antigens since infected animals are able to develop a T cell response to the mitogen Concanavalin A (Con-A) and, after sensitization, to the antigens keyhole limpet haemocyanin (KLH) and human serum albumin (DNP-HSA). In the present investigations we studied the role played by infected macrophages in the development of the cellular unresponsiveness present in visceral leishmaniasis. Adherent spleen cells from infected hamsters were unable to present L. donovani antigens to antigen specific T cells, however they were able to present KLH. Conversely, T cells from infected animals did not respond to parasite antigens even when these antigens were presented by normal syngeneic macrophages. Interestingly, lymphocytes from inguinal lymph nodes of infected animals sensitized in their foot pad with parasite antigens proliferated well when stimulated in vitro with L. donovani antigens. These results suggest that the defect in the cellular immune response of the L. donovani infected hamsters is a consequence of a selective inability of their antigen presenting cells to process and present parasite antigens to T cells.     

Cellular responses to phase fractions of Trypanosoma lewisi

Infections by Trypanosoma lewisi are characterized by hyporesponsiveness of the immune system during the early phase of parasitaemia. Blastogenic response of normal rat spleen cells to amphiphilic and hydrophilic components of Triton X-114 solubilized epimastigote forms of T. lewisi which characterizes the early phase of infection showed that suppression of responses to mitogens Concanavalin A (Con-A) and lipopolysaccharide (LPS) occurred exclusively with the amphiphilic fraction that consists of integral surface membrane constituents. The Con-A-induced suppression by the amphiphilic constituents was ablated by addition of exogenous IL-2 or by the removal of the adherent cell population in the cultures. This suggests that the integral surface membrane components play an important regulatory role in infections with Trypanosoma lewisi, through complex mechanisms that probably involve the B cells and suppressor macrophages; the suppressor macrophages probably produce a suppressor factor that inhibits the proliferation of T helper cells and subsequently the production of interleukin-2.     

Studies on the effect of the anti-phagocytic agent cytochalasin B on Leishmania-macrophage interaction.

Mouse peritoneal exudate cells were cultured on coverslips in Eagle's Basal Essential Medium. The adhering cells were infected with promastigotes of three different species of Leishmania. After 8 h incubation, the macrophages were fixed and stained, and a total of one hundred cells were counted. The rates of infection of macrophages were respectively 53.5 +/- 5% for L. enriettii, 52.3 +/- 5% for L. donovani and 11.7 +/- 2% for L. tropica. When cytochalasin B at concentrations of 2.5, 5 and 10 microgram/ml and Leishmania promastigotes were added to the adhering cells at the same time, the drug did not have any effect on the uptake of the organisms by the macrophages. However, when the cells were treated for a 2-h period with the drug and then were infected with the promastigotes, only 1-2% of the cells were infected. On the other hand, when cytochalasin B-treated cells which had lost their phagocytic ability were washed and then were infected with the promastigotes, some degree of cellular infection was observed. It was concluded that infection of mouse p.e.c. by three different species of Leishmania which were used in our study was by phagocytosis rather than active penetration of the organisms into the cells. It was also of interest to note that although our outbred strain of mice gets infected easily with L. tropica, the p.e.c. of these animals phagocytosed L. tropica with least efficiency in comparison with L. donovani and L. enriettii.     

杜氏利什曼原虫蛋白磷酸酶-2C的基因克隆化与序列分析

目的　克隆杜氏利什曼原虫 (Leishmaniadonovani,Ld) 1S株蛋白磷酸酶 2C(PP2C)的编码基因 ,为应用这种编码T细胞抗原的基因进行基因疫苗研究奠定基础。方法　体外培养杜氏利什曼原虫 1S株无鞭毛体 ,常规方法从虫体提取制备基因组DNA。以夏科氏利什曼原虫 (Leishmaniachagasi,Lc)的PP2C基因序列为参照 ,设计并合成利什曼原虫PP2C基因序列特异性的引物。结果　以杜氏利什曼原虫的基因组为模板 ,利用多聚酶链反应 (PCR)技术 ,扩增获得了杜氏利什曼原虫PP2C的全长编码基因。基因序列测定结果表明 ,杜氏利什曼原虫 1S株PP2C基因序列长度为 1317bp ,开放读码框架由 12 2 1bp组成 ,编码产物为 40 6个氨基酸残基。获得的杜氏利什曼原虫 1S株的PP2C基因与来源于夏科氏利什曼原虫的PP2C氨基酸残基序列的同源性为 95 % (387/ 40 6 )。结论　本研究克隆了杜氏利什曼原虫的PP2C基因 ,为应用诱导T细胞免疫应答抗原的编码基因进行杜氏利什曼原虫的基因疫苗研究奠定了基础     

The activity of nitrofurazone and furazolidone against Leishmania donovani, L. major and L. enriettii in vitro and in vivo

Furazolidone and nitrofurazone showed in vitro activity against amastigotes of Leishmania donovani, L. enriettii and L. major in macrophages, at concentrations which were also toxic to the macrophages. A low grade of activity was observed against L. donovani infections in BALB/c mice by furazolidone but not with nitrofurazone. Nitrofurazone, in two concentrations, was not active when applied to the lesions of cutaneous leishmaniasis due to L. enriettii (guinea-pig infection) or L. major strain P (BALB/c mouse infection). After systemic administration to BALB/c mice infected with L. major strain JISH 252 clone 1, low-grade activity was observed at the highest level tested.